Transgenic expression of malaria surface antigens under the control of phaseolin promoter.

Chan Wan Lui Wendy.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2004.
Includes bibliographical references (leaves 158-162).
s in English and Chinese.
Acknowledgements --- p.iii
p.v
List of Abbreviations --- p.ix
List of Figures --- p.xii
List of Tables --- p.xvi
Table of Contents --- p.xvii
Chapter Chapter 1 --- General Introduction --- p.1
Chapter Chapter 2 --- Literature review --- p.3
Chapter 2.1 --- Malaria --- p.3
Chapter 2.2 --- History of malaria --- p.4
Chapter 2.3 --- Malaria parasites --- p.4
Chapter 2.4 --- Life cycle --- p.5
Chapter 2.5 --- Potential use of malaria vaccine --- p.6
Chapter 2.6 --- Merozoite surface protein 1 (MSP1) --- p.7
Chapter 2.7 --- Potential use of MSPl --- p.8
Chapter 2.8 --- Significance of MSPl C-terminal fragments --- p.9
Chapter 2.8.1 --- Significance of MSP142 --- p.9
Chapter 2.8.2 --- Significance of MSP119 --- p.11
Chapter 2.9 --- Production of MSPl C-terminal fragments --- p.12
Chapter 2.10 --- Plants as bioreactors --- p.12
Chapter 2.11 --- Expression of MSPl C-terminal fragments in transgenic plants --- p.14
Chapter 2.12 --- Phaseolin and its sorting signal --- p.19
Chapter 2.13 --- Protein targeting signals --- p.20
Chapter Chapter 3 --- Material and methods --- p.23
Chapter 3.1 --- Introduction --- p.23
Chapter 3.2 --- Chemical and enzymes --- p.23
Chapter 3.3 --- Cloning --- p.24
Chapter 3.3.1 --- MSP142 and MSP119 constructs --- p.24
Chapter 3.3.2 --- Protein targeting fusion constructs --- p.24
Chapter 3.3.3 --- GUS fusion Constructs --- p.30
Chapter (a) --- Particle bombardment --- p.30
Chapter (b) --- GUS fusion constructs for plant transformation --- p.32
Chapter (c) --- Modified GUS fusion constructs --- p.38
Chapter 3.4 --- Cloning of chimeric gene into Agrobacterium binary vector --- p.39
Chapter 3.4.1 --- Cloning of pSUNl --- p.40
Chapter 3.4.2 --- Primer sequence --- p.45
Chapter 3.5 --- Bacterial strains --- p.46
Chapter 3.6 --- Particle bombardment --- p.46
Chapter 3.6.1 --- Plant materials --- p.46
Chapter 3.6.2 --- Microcarrier preparation and coating DNA onto microcarrier --- p.46
Chapter 3.6.3 --- GUS assay --- p.48
Chapter 3.7 --- Transgenic expression in Arabidopsis thaliana --- p.49
Chapter 3.7.1 --- Plant materials --- p.49
Chapter 3.7.2 --- Agrobacterium transformation --- p.49
Chapter 3.7.3 --- Vacuum infiltration Arabidopsis transformation --- p.49
Chapter 3.7.4 --- Selection of successful transformants --- p.50
Chapter 3.7.5 --- Selection for homozygous plants --- p.51
Chapter 3.8 --- Transgenic expression in tobacco --- p.51
Chapter 3.8.1 --- Plant materials --- p.51
Chapter 3.8.2 --- Agrobacterium transformation --- p.52
Chapter 3.8.2.1 --- Preparation of Agrobacterium tumefaciens LBA4401 competent cells --- p.52
Chapter 3.8.3 --- Leaf discs method for tobacco transformation --- p.53
Chapter 3.8.4 --- GUS staining --- p.54
Chapter 3.9 --- DNA analysis --- p.55
Chapter 3.9.1 --- Genomic DNA extraction --- p.55
Chapter 3.9.2 --- Genomic PCR --- p.55
Chapter 3.9.3 --- Southern blot --- p.55
Chapter 3.10 --- RNA analysis --- p.56
Chapter 3.10.1 --- RNA extraction --- p.56
Chapter 3.10.2 --- Northern blot --- p.56
Chapter 3.11 --- Protein analysis --- p.57
Chapter 3.11.1 --- Protein extraction --- p.57
Chapter 3.11.2 --- Western blot --- p.58
Chapter 3.11.3 --- Western blot analysis --- p.58
Chapter Chapter 4 --- Results --- p.60
Chapter 4.1 --- Transient assay of gene expression of MSP142 and MSPl19 --- p.60
Chapter 4.1.1 --- Construction of the GUS fusion constructs --- p.60
Chapter 4.1.2 --- Particle Bombardment --- p.63
Chapter 4.2 --- Transgenic analysis of MSP142 and MSPl19 expression --- p.70
Chapter 4.2.1 --- MSPl42 and MSPl19 constructs and transformation --- p.70
Chapter 4.2.2 --- Selection of transgenic plants --- p.71
Chapter 4.2.3 --- Southern analysis --- p.75
Chapter 4.2.4 --- Northern analysis --- p.77
Chapter 4.2.5 --- Western analysis --- p.79
Chapter 4.3 --- Expression of the protein-targeting and GUS fused modified MSP1 constructs --- p.81
Chapter 4.3.1 --- Construction of the fusion constructs --- p.81
Chapter (A) --- Protein-targeting constructs --- p.81
Chapter (B) --- GUS fusion constructs --- p.90
Chapter B1. --- Constructs for transient assay --- p.90
Chapter B2. --- Modification of GUS sequence --- p.96
Chapter B3. --- Constructs for tobacco transformation --- p.100
Chapter 4.4 --- Transient assay of GUS fused MP42 and MP19 constructs by particle Bombardment --- p.107
Chapter 4.4.1 --- The GUS fusion constructs --- p.107
Chapter 4.4.2 --- Modification of GUS --- p.112
Chapter 4.5 --- Generation of transgenic tobacco --- p.116
Chapter 4.6 --- Southern analysis --- p.120
Chapter 4.7 --- Northern analysis --- p.126
Chapter (A) --- Protein-targeting constructs --- p.126
Chapter (B) --- GUS fusion constructs --- p.130
Chapter 4.8 --- Western analysis --- p.133
Chapter (A) --- Protein-targeting constructs --- p.133
Chapter (B) --- GUS fusion constructs --- p.139
Chapter Chapter 5 --- Discussion --- p.146
Chapter Chapter 6 --- Conclusion --- p.157
References --- p.158
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